Polycrystalline form of dehydrophenylahistin-like compound, and manufacturing and purification method and application thereof

ABSTRACT

A polycrystalline form of a dehydrophenylahistin-like compound, and a manufacturing and purification method and application thereof A (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione monohydrate crystal and a (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione monohydrate crystal are more competitive crystalline forms with stable quality. The manufacturing and purification method is simple and easy to operate, and can effectively control the generation of a trans-isomer contaminant to obtain a high purity product. The polycrystalline form of the dehydrophenylahistin-like compound has a certain value in an application for manufacturing an antitumor pharmaceutical product.

TECHNICAL FIELD

The present invention belongs to the field of pharmaceuticalchemistryand relates to polymorphs of dehydrophenylahistin-likecompounds and method for preparation and purification thereof and usethereof.

BACKGROUND

(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionebelongs to dehydrophenylahistin-like compounds and has a formula of:

Said compound is also known as Plinabulin (KPU-2, NPI-2358) and isdeveloped by Nereus Pharmaceuticals Inc (US), and is a syntheticderivative of low molecular cyclo-dipeptide phenylahistin or halimidefrom marine Aspergillus, and is a tubulin-binding agent. Plinabulin canbind on the neighborhood of colchicine-binding site in tubulin and acton the cell to stop cell mitosis in its early stages which in turninduces cell death. At the same time, plinabulin also inhibitsmicrotubule formation and migration of endothelial cells and MM cells,leading to dysfunction of tumor vasculature. Currently, plinabulin, as adrug candidate, has completed its Phase II clinical trials in the UnitedStates and is under Phase III clinical trials in China and the UnitedStates.

(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1-H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione belongs to deuterated dehydrophenylahistin-like compoundsand has a formula of:

Said compound is a novel tubulin-binding agent obtained by structuralmodification using plinabulin as a lead compound. It has good antitumoractivity and can overcome the drug tolerance of paclitaxel. Itselectively acts on the neighborhood of colchicine-binding site inendothelial tubulin, inhibits the polymerization of tubulin, blocks offmicrotubule formation, stops cell mitosis in its early stages andthereby induces cell death. At the same time, said compound alsoinhibits neovascularization, blocks cancer cell feeding, and thereforesynergistically inhibits rapid proliferation of cancer cells.

Studies show that the IC50 of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione is lower than that of its analog plinabulin, butits water solubility is only 39.90 ng/mL, which seriously limits itsclinical application. The inventors of the present invention has figuredout the key techniques for preparing(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionethrough previous laborious work, for which a patent named DeuteratedDehydrophenylahistin-like Compouds And Preparation Method Thereof AndUse Thereof In Preparation Of Anti-Tumor Drugs having a patentapplication number: 201510293269.8 has been filed for Chinese inventionpatent and PCT application.

Currently formulation of plinabulin applied for clinical trials is aconcentrated injection solution with a solubility enhancer, being aninjection, the quality and stability of plinabulin as a solid API arecritically required, especially it is essential to obtain a crystallineform with high purity and good stability. Drug polymorphism is a commonphenomenon in drug development. Different crystalline forms of the samedrug molecule may have significant differences in appearance, meltingpoint, solubility, dissolution rate, bioavailability, etc., whichdirectly affect drug stability, bioavailability and therapeutic effects.Therefore, studying and screening plinabulin polymorphs are of greatimportance.

Currently there are no reports on the crystalline forms of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneand their preparation methods.

At the same time, both(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneand deuterated dehydrophenylahistin-like compound(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionehave strong photosensitivity, especially in solution, leading to thegeneration of isomers(3E,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneand(3E,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneduring their preparation processes. As the above-mentioned isomers arenot easy to be removed, it poses a problem for subsequent formulationand especially has impact on new drug application process as well asdrug safety in clinical application.

Therefore, preparation of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewith high purity is of great significance. Moreover, currently reportedmethods for preparation and purification of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneare complex, wherein purification of some intermediates through columnchromatography limits its industrialized production, so it is of greatimportance to set up a suitable industrialized preparation method.

Summary of the Invention

The object of the present invention is to provide polymorphs ofdehydrophenylahistin-like compounds, method for preparation andpurification thereof and use thereof.

In order to achieve said objects of the present invention, the followingtechnical solutions are provided:

In one aspect, the present invention provides a crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneas shown in Formula (I), and said crystalline form has at least threeX-ray powder diffraction characteristic peaks at 2θ diffraction angle of8.415°±0.2°, 11.512°±0.2°, 14.824 °±0.2°, 17.087°±0.2°, 17.278°±0.2°,19.461°±0.2°, 21.350°±0.2°, 22.344°±0.2° or 27.621°±0.2;

preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.415°±0.2°,11.512°±0.2°, 14.824°±0.2°, 17.278°±0.2°, 19.461°±0.2°, 21.350°±0.2°,22.344±0.2° and 27.621°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 20 diffraction angle of 8.415°±0.2°,11.512°±0.2°, 14.824°±0.2°, 17.087°±0.2°, 17.278°±0.2°, 19.461°±0.2°,21.350°±0.2°, 22.344°±0.2° and 27.621°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.415°±0.2°,11.512°±0.2°, 12.271°±0.2°, 13.126°±0.2°, 13.618°±0.2°, 14.824°±0.2°,16.011°±0.2°, 16.282°±0.2°, 17,087°±0.2°, 17.278°±0.2°, 17.608°±0.2°,18.134°±0.2°, 18.408°±0.2°, 19.461°±0.2°, 19.735°±0.2°, 20.745°±0.2°,21.350°±0.2°, 22.344°±0.2°, 23.198°±0.2°, 24.874°±0.2°, 25.168°±0.2°,26.997°±0.2°, 27.621°±0.2° and 28.479°±0.2°;

most preferably, the X-ray powder diffraction pattern of saidcrystalline form is consistent with FIG. 1.

Said crystalline form is named as α-crystalline form, the method for itspreparation comprises the following steps:

cyclizing ethyl isocyanoacetate with trimethylacetic anhydride underalkaline condition to give 5-(tert-butyl)oxazole-4-ethyl formate;

converting 5-(tert-butyl)oxazole-4-ethyl formate to an imidazole ringunder heating in formamide, followed by reduction by lithium aluminumhydride, oxidization by manganese dioxide, reduction by deuteratedsodium borohydride, and reoxidiztion by manganese dioxide to give5-(tert-butyl)-1H-imidazol-4-deuteroformaldehyde; dissolving glycineanhydride in acetic anhydride to give 1,4-diacetylpiperazine-2,5-dione;

condensing 5-(tert-butyl)-1H-imidazol-4-deuteroformaldehyde with1,4-diacetylpiperazine-2,5-dione under alkaline condition, followed bycondensation with benzaldehyde and purification to give an α-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneas a pale-yellow crystalline powder.

In another aspect, the present invention also provides a crystallineform of (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione, and saidcrystalline form has at least three X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 7.629°±0.2°,8.052°±0.2°, 12.967±0.2°, 15.327°±0.2°, 16.195°±0.2°, 23.194°±0.2°,23.760°±0.2°, 24.129°±0.2°, 24.419°±0.2°, 26.465°±0.2° or 29.213°±0.2°;

preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 20 diffraction angle of 7.629°±0.2°,8.052°±0.2°, 12.967°±0.2°, 15.327°+0.2°, 16.195°±0.2°, 23.194°±0.2°,23.760°±0.2°, 24.129°±0.2°, 24.419°±0.2°, 26.465°±0.2°and 29.213°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 20 diffraction angle of 7.629°±0.2°,8.052°±0.2°, 8.958°±0.2°, 12.967°±0.2°, 15.327±0.2°, 16.195°±0.2°,16.606°±0.2°, 17.410°±0.2° , 23.194°±0.2°, 23.760°±0.2°, 24.129°±0.2°,24.419°±0.2°, 25.256°±0.2°, 26.465°±0.2° and 29.213°±0.2°;

most preferably, the X-ray powder diffraction pattern of saidcrystalline form is consistent with FIG. 2.

Said crystalline form is named as β-crystalline form, and itspreparation method comprises the following steps: dissolving(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-yl)deuteromethylene]piperazine-2,5-dionecompletely till saturation using a solvent at 15° C. to 100° C.,standing and cooling to 20° C.-30° C. in the absence of light, andnaturally volatilizing the solvent to give a β-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione,and said solvent is methanol, ethanol, isopropanol, acetone or water, ora mixed solvent of two or more thereof;

preferably, said solvent is a mixed solvent of water and methanol with avolume ratio of V_(water):V_(methanol) ranging from 1:100© to 3:100, andthe heating temperature for dissolution ranges from 60° C. to 80° C.

Further, said prepared n-crystal line form is a single crystal in whichone molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis combined with one molecule of methanol, and its appearance is cubic.It is analyzed by X-ray single crystal diffraction to be a monoclinicsingle crystal having a. space group of P2(1)/n, its analyticalstructure is consistent with FIG. 3, and its melting point is 263.6° C.to 264.4° C.

In still another aspect, the present invention also provides acrystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione,and said crystalline form has at least three X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.075°±0.2°,12.986°±0.2°, 16.217°±0.2°, 19.709°+0.2° or 24.441°±0.2°;

preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 20 diffraction angle of 8.075°±0.2°,12.986°±0.2°, 16.217+0.2°, 19.709°±0.2° and 24.441°±0.2°;

more preferably, the X-ray powder diffraction pattern of saidcrystalline form is consistent with FIG. 4.

Said crystalline form is named as γ-crystalline form, and itspreparation method comprises the following steps: dissolving(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionecompletely till saturation at 15° C. to 100° C. using methanol, ethanol,isopropanol, acetone or water or a mixed solvent of at least twothereof, standing and cooling to 15° C.-20° C. in the absence of light,and naturally volatilizing the solvent to give a γ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione.

Further, said solvent is a mixture of water and methanol with a volumeratio of V_(water):V_(methanol) ranging from 3:100 to 1:5, and theheating temperature for dissolution is 25° C. to 50° C.

Further, said prepared γ-crystalline form is a single crystal in whichone molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis combined with two molecules of methanol, and its appearance isdiamond-shaped. It is analyzed by X-ray single crystal diffraction to bea triclinic single crystal having a space group of P-1, its analyticalstructure is consistent with FIG. 5, and its melting point is 263.6°C.-265.1° C.

In still another aspect, the present invention also provides acrystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionemonohydrate, and said crystalline form has at least three X-ray powderdiffraction characteristic peaks at 2θ diffraction angle of 8.075°±0.2°, 12.988°±0.2°, 16.201°±0.2°, 17.545°±0.2°19.084°±0.2°,19.724°±0.2°, 23.710°±0.2°, 24.422°±0.2°, 26.485°±0.2° or 29.234°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 20θ diffraction angle of 8.075°±0.2°,12.988°±0.2°, 16.201°±0.2°, 19.084°±0.2°, 19.724°±0.2°, 24.422°±0.2° and29.234°±0.2;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.075°±0.2°,12.988°±0.2°, 16.201°±0.2°, 17.545°±0.2°, 19.084°±0.2°, 19.724°±0.2°,23.710°±0.2°±24.422°±0.2°, 26.485°±0.2° and 29.234°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.075°±0.2°,9.145°±0.2°, 12.988°±0.7°, 14.740°±0.2°, 16.201°±0.2°, 17.545°±0.2°,18.367°±0.2°, 19.084°±0.2°, 19.724°±0.2°, 22.781°±0.2°, 23.710°±0.2°,24.422°±0.2°, 25.279°±0.2°, 26.485°±0.2°, 27.867°±0.2° and 29.234°±0.2°;

most preferably, the X-ray powder diffraction pattern of saidcrystalline form is consistent with FIG. 6.

Said crystalline form is named as δ-crystalline from, and itspreparation method comprises the following steps: recrystallizing(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionein a mixed solvent of water and an organic solvent, wherein said organicsolvent is selected from at least one of an alkane saturated alcohol, anunsaturated alcohol, an alkane saturated amine and an unsaturated amine,cooling and stirring to precipitate to give a δ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione,and said crystalline form is a monohydrate of(3Z,6Z)3-benzylidene-6-[(5terrt-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione,and its melting point is 263.5° C. to 266.5° C.

Further, said organic solvent is preferably methanol, ethanol,isopropanol or acetone; said mixed solvent comprises water andisopropanol with a volume ratio of V_(water):V_(isopropanol) rangingfrom 1:100 to 10:1, said mixed solvent comprises water and methanol witha volume ratio of V_(water):V_(methanol) ranging from 1:100 to 10:1, andsaid mixed solvent comprises water and ethanol with a volume ratio ofV_(water):V_(ethanol) ranging from 1:100 to 10:1.

Further, placing the crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionein a reaction vessel, in the absence of light, adding isopropanol assolvent, heating to dissolve completely, then adding water with a volumeratio of V_(water):V_(isopropanol) ranging from 1:100 to 1:1, standingat −10° C. to 30° C., stirring and cooling to precipitate, followed bysuction filtering, washing and drying to give a pale-yellow solid whichis(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionemonohydrate with high purity having a trans-isomer content of less than0.1%, i.e. δ-crystalline form.

Further, said volume ratio of isopropanol to water is V_(isopropanol):V_(water)=5:2, and the precipitation temperature preferably is −5° C. to10° C.

Method of preparing a δ-crystalline single crystal of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionecomprises the following steps: dissolving(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionecompletely till saturation at 25° C. to 100° C. in methanol, ethanol,isopropanol, acetone or water or a mixed solvent of at least twothereof, in the absence of light, standing and cooling to 10° C. to 50°C., then naturally volatilizing the solvent to give a δ-crystallinesingle crystal of (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione.

Further, said prepared δ-crystalline form is a single crystal in whichone molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1-H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis combined with one molecule of water, and its appearance isneedle-like. It is analyzed by X-ray single crystal diffraction to be amonoclinic single crystal having a space group of P2(1)/n, itsanalytical structure is consistent with FIG. 7, and its melting point is263° C. to 267° C.

In still another aspect, the present invention also provides acrystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazo1-yl)deuteromethylene]piperazine-2,5-dionemonoisopropylate, named as c-crystalline form, and its preparationmethod comprises the following steps: dissolving(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionecompletely till saturation at 10° C. to 100° C. in a mixed solvent ofisopropanol and water, in the absence of light, standing and cooling to10° C. to 50° C., then naturally^(,) volatilizing the solvent to give aδ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione.

Further, the used solvent comprises purified water and isopropanol witha volume ratio of V_(water):V_(isopropanol) ranging from 1:200 to 1:10.

Further, said prepared ϵ-crystalline form is a single crystal in whichone molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazoi-4-yl)deuteromethylene]piperazine-2,5-dioneis combined with one molecule of isopropanol, and its appearance isdiamond-shaped. It is analyzed by X-ray single crystal diffraction to bea monoclinic single crystal having a space group of P2(1)/n, itsanalytical structure is consistent with FIG. 9, and its melting point is264.1° C. to 264.7° C.

In still another aspect, the present invention also provides a use ofthe aforementioned crystalline forms of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol4-yl)deuteromethylene]piperazine-2,5-dionefor preparation of antitumor drugs.

In still another aspect, the present invention also provides acrystalline form of (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4yl)methylene]piperazine-2,5-dioneas shown in Formula (II), and said crystalline form has at least threeX-ray powder diffraction characteristic peaks at 20 diffraction angle of7.670±0.2°, 9.069°±0.2°, 15.383°±0.2°, 16.668°±0.2°, 17.468°±0.2°,18.109°±0.2°, 19.960°±0.2°, 23.307°±0.2°, 23.836°±0.2°, 24.462°±0.2°,28.046°±0.2° or 28.827°±0.2°;

Preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 7.670°±0.2°,9.069°±0.2°, 15.383°±0.2°, 16.668°±0.2° and 23.836°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 7.670°±0.2°,9.069°±0.2°, 15.383°±0.2°, 16.668°±0.2°, 17.468°±0.2°, 18.109°±0.2°,19.960°±0.2°, 23.307±0.2°, 23.836°±0.2°, 24.462°±0.2°, 28.046°±0.2° and28.827±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 7.356°±0.2°, 7.670°,40.2°, 8.097°±0.2°, 9.069°±0.2°, 12.032°±0.2°, 12.500°±0.2°,13.063°±0.2°, 16.241°±0.2°, 16.668°±0.2°, 17.468°±0.2°, 18.109°±0.2°,18.694°±0.2°, 19.960°±0.2°, 23.307°±0.2°, 23.836°±0.2°, 24.462°±0.2°,28.046°±0.2°, 28.827°±0.2° and 30.226°±0.2°;

most preferably, the X-ray powder diffraction pattern of saidcrystalline form is consistent with FIG. 11.

Said crystalline form is named as b-crystalline form, and itspreparation method comprises the following steps: dissolving(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionee completely till saturation at 40° C. to 100° C. in a mixed solvent ofwater and methanol with a volume ratio of V_(water):V_(methanol) rangingfrom 1:1000 to 3:100, in the absence of light, standing and cooling to15° C. to 30° C., then naturally volatilizing the solvent to give ab-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione.

Further, said mixed solvent comprises water and methanol with a volumeratio of V_(water) :V_(methanol) ranging from 1:1000 to 1:100, and thetemperature for complete dissolution is 60° C. to 80° C.

Further, the prepared b-crystalline form is a single crystal in whichone molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneis combined with one molecule of methanol, and its appearance is cubic.It is analyzed by X-ray single crystal diffraction to be monoclinichaving a space group of P2(1)/n, its analytical structure is consistentwith FIG. 12, and its melting point is 264.0° C. to 264.9° C.

In still another aspect, the present invention also provides acrystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione,and said crystalline form has at least three X-ray powder diffractioncharacteristic peaks at 2θ diffractionangle of 7.918°±0.2°, 9.168°±0.2°,12.985°±0.2°, 18.382°±0.2° 18.616°±0.2°, 23.367°±0.2°, 25.203°±0.2° or27.771°±0.2°;

preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 7.918°±0.2°,9.168°±0.2°, 18.382° and 18.616°±0.2°;

more preferably, said crystal form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 7.918°±0.2°,9.168°±0.2°, 12.014°±0.2°, 12.985°±0.2°, 18.382°±0.2°, 18.616°±0.2°,23.367°±0.2°, 25.203°±0.2° and 27.771°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 7.918°±0.2°,9.168°±0.2°, 9.905°±0.2°, 12.014°±0.2°, 12.985°±0.2′, 14,970″±0.2″,15.8⁷3°±0.2°, 18.382°±0.2°, 18.616°±0,2″, 19.081°±0.2°, 19.881°±0.2°,22.862°±0.2°, 23.367°±0.2°, 23.719°±0.2°, 24.073°±0.2°, 25.203°±0.2°,26.447°±0.2°, 27.771°±0.2° and 37.787°±0.2°;

most preferably, the X-ray powder diffraction pattern of saidcrystalline form is consistent with FIG. 13.

Said crystalline form is named as c-crystalline form, and itspreparation method comprises the following steps: dissolving(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionecompletely at 60° C. to 100° C. in a mixed solvent of water and methanolwith a volume ratio of V_(water):V_(methanol) ranging from 3:100 to 1:5,in the absence of light, standing and cooling to 15° C. to 30° C., thennaturally volatilizing the solvent to obtain a c-crystalline form of(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione.

Further, the volume ratio of water to methanol : V_(water):V_(methanol)in said mixed solvent ranges from 3:100 to 1:20.

Further, the prepared c-crystalline form is a single crystal in whichone molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneis combined with one molecule of methanol, and its appearance isneedle-like. It is analyzed by X-ray single crystal diffraction to bemonoclinic with a space group of P2(1)/n, its analytical structure isconsistent with FIG. 14, and its melting point is 263.2° C. to 264.0° C.

In still another aspect, the present invention also provides acrystalline form of(3Z,6Z)3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione,and said crystalline is a(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionemonohydrate; preferably said crystalline form has at least three X-raypowder diffraction characteristic peaks at 2θ diffraction angle of8.073°±0.2°, 13.005°±0.2°, 17.544°±0.2°, 18.382°±0.2°, 19.082°±0.2°,19.707°±0.2°, 22.766°±0.2°, 23.759°±0.2°, 24.438°±0.2°, 25.277°±0.2°,26.486°±0.2° or 29.234°±0.2°;

preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.073°±0.2°,13.005°±0.2°, 19.082°±0.2° 19.707°±0.2° and 23.759°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.073°±0.2°,13.005°±0.2°, 17.544°±0.2°, 18.382°±0.2°, 19.082°±0.2°, 19.707±0.2°,22.766°±0.2°, 23.759°±0.2°, 24.438°±0.2°, 25.277°±0.2°, 26.486°±0.2° and29.234°±0.2°;

more preferably, said crystalline form has an X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.073°±0.2°,9.146°±0.2°, 13.005°±0.2°, 14.740°±0.2°, 16.184°±0.2°, 17.544°±0.2°,18.382°±0.2°, 19.082°±0.2°, 19.707°±0.2°, 22.766°±0.2°, 23.759°±0.2°,24.438°±0.2°, 25.277°±0.2°, 26.486°±0.2°, 27.883°±0.2° and 29.234°±0.2°;

most preferably, the X-ray powder diffraction pattern of saidcrystalline form is consistent with FIG. 15.

Said crystalline form is named as d-crystalline form, and itspreparation method comprises the following steps: recrystallizing(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionein a mixed solvent of water and an organic solvent, wherein said organicsolvent is selected from at least one of an alkane saturated alcohol, anunsaturated alcohol, an alkane saturated amine or an unsaturated amine,cooling and stirring to precipitate to give a d-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione,and said crystalline form is a monohydrate, and its melting point is263.5° C. to 266.5° C.

Further, the volume ratio of water to isopropanolV_(water):V_(isopropanol) in said mixed solvent ranges from 1:100 to99:100.

Further, the volume ratio of water to isopropanolV_(water):V_(isoproponal) in said mixed solvent ranges from 1:10 to9:10.

Further, placing the crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionein a reaction vessel, in the absence of light, adding isopropanol assolvent, dissolving completely under heating, then adding water with avolume ratio of V_(isopropanol):V_(water) ranging from 1:10 to 100:1,standing at −15° C. to 30° C., stirring and cooling to precipitate,followed by suction filtering, washing and drying to give a pale-yellowsolid which is a monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewith high purity having a trans-isomer content of less than 0.1%.

Further, said volume ratio of isopropanol to water isV_(isopropanol):V_(water)=5:1.

The present invention provides a method of preparing a d-crystallinesingle crystal of(3Z,6Z)-3-benzylidene-6[(5tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione,comprising the following steps: dissolving(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionecompletely till saturation at 25° C. to 100° C. in methanol, ethanol,isopropanol, acetone or water or a mixed solvent of at least twothereof, in the absence of light, standing and cooling to 10° C. to 50°C., then naturally volatilizing the solvent to give a d-crystalline formof(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione.

Further, the used solvent comprises water and isopropanol with a volumeratio of V_(water):V_(isopropanol) ranging from 1:200 to 7:20.

Further, the used solvent comprises water and ethanol with a volumeratio of V_(water):V_(ethanol) ranging from 1:200 to 1:2.

Said d-crystalline form prepared according to aforementioned method is asingle crystal in which one molecule of(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneis combined with one molecule of water, and its appearance is longcolumnar or needle-like. It is analyzed by X-ray single crystaldiffraction to be a monoclinic single crystal with a space group ofP2(1)/n, its analytical structure is consistent with FIG. 17, and itsmelting point is 263° C. to 267° C.

In still another aspect, the present invention also provides a method ofpreparing and purifying a dehydrophenylahistin-like compound with highpurity, and said method comprises the following steps: placing the crudeproduct of a dehydrophenylahistin-like compound in a reaction vessel, inthe absence of light, adding isopropanol or methanol or ethanol orn-butanol under heating condition till complete dissolution of saidcompound, then adding water resulting in no crystalline precipitation,standing at 15° C. to 30° C., stirring and cooling to precipitate,followed by suction filtering, washing and drying to give adehydrophenylahistin-like compound with high-purity.

Further, said dehydrophenylahistin-like compound has a structurerepresented by Formula (III):

wherein R₁ is a mono-substituted to penta-substituted substituent groupon the benzene ring, which is independently selected from hydrogen,deuterium, 3-benzoylphenyl, 3-(4-methoxybenzoyl)phenyl,3-(4-fluorobenzoyl)phenyl, halogen, hydroxy, methoxy, amino, phenyl,aminomethylphenyl, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl,arylalkyl, heterocycloarylalkyl, C1-C24 acyl, C1-C24 alkoxy, carboxy,carboxylate, acylamino, N-monosubstituted or N,N-disubstitutedacylamino, sulfo, sulphonate, sulphonylamino, N-substitutedsulphonylamino, alkoxy, arylalkoxy, alkylsulfanyl, cyano, amino,substituted amino, nitro, cycloalkyl, cycloalkenyl, aryl, substitutedaryl, heterocycloaryl, aryloxy, aroyl, epoxy group, cycloacyl,arylsulfenyl, arylsulfonyl;

R₂ is hydrogen or deuterium, and R₃ is hydrogen or deuterium;

X₁ is oxygen or sulfur, and X₂ is oxygen or sulfur;

X₃ is —NH, oxygen or sulfur;

further, when said dehydrophenylahistin-like compound is(3,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione,in particular, when preparing a (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione monohydrate withhigh purity, the crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis placed in a reaction vessel, in the absence of light, isopropanol isadded as solvent, completely dissolving under heating, water is addedthen with the resulting volume ratio of V_(isopropanol):V_(water)ranging from 1:5 to 100:1, standing at −10° C. to 30° C., stirring andcooling to precipitate, suction filtered, washed and dried to give apale-yellow solid which is a monohydrate of (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionewith high purity having a trans-isomer content of less than 0.1%.

Further, said volume ratio of isopropanol to water isV_(isopropanol):V_(water)=5:2.

Alternatively, said dehydrophenylahistin-like compound is(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione, and method of preparing the crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine -2,5-dione compound is as follows:

cyclizing ethyl isocyanoacetate with trirnethylacetic anhydride underalkaline condition to give 5-(tert-butyl)oxazole-4-ethyl formate;

then converting 5-(tert-butyl)oxazole-4-ethyl formate into an imidazolering under heating in formamide, followed by reduction by lithiumaluminum hydride, and oxidization by manganese dioxide to give5-(tert-butyl)-1H-imidazole-4-formaldehyde; dissolving glycine anhydridein acetic anhydride to give 1,4-diacetylpiperazine-2,5-dione;

condensing 5-(tert-butyl)-1H-imidazole-4-formaldehyde with1,4-diacetylpiperazine-2,5-dione under alkaline condition, then furthercondensing with benzaldehyde to give a crude product of(3Z,6Z-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione.

Further, placing the crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionein a reaction vessel, in the absence of light, adding isopropanol assolvent and dissolving completely under heating, adding water with avolume ratio of V_(isopropanol):V_(water) ranging from 1:10 to 100:1,standing at −15° C. to 30° C., stirring and cooling to precipitate,followed by suction filtering, washing and drying to give a pale-yellowsolid which is a(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionemonohydrate with high purity having a trans-isomer content of less than0:1%.

Further, said volume ratio of isopropanol to water isV_(isopropanol):V_(water)=5:2.

Further, said 5-(tert-butyl)-1H-imidazole-4-ethyl formate is purified bythe following steps: reacting 5-(tert-butyl)-1H-oxazole-4-ethyl formatewith formamide under heating, after the reaction is completed, coolingthe solution, extracting with sodium carbonate and petroleum ether,separating the impurities in petroleum ether phase, extracting withethyl acetate, combining the organic phases, washing with water, dryingand concentrating under reduced pressure to give a concentrated solutionwhich, while being warm, is poured into water, stirred and pulpifiedtill a solid precipitates, followed by suction filtering, washing anddrying in vacuo to give a purified 5-(tert-butyl)-1H-imidazole-4-ethylformate as mentioned above.

Further, said I,4-diacetylpiperazine-2,5-dione is purified by thefollowing steps: refluxing and reacting of glycine anhydride with aceticanhydride, after the reaction is completed, cooling the resultingsolution to room temperature, concentrating under reduced pressure toevaporate the solvent, dissolving the concentrate in dichloromethane,filtering through diatomite, concentrating under reduced pressure toremove dichloromethane, recrystallizing in ethyl acetate, precipitatingat low temperature, filtering and drying to give a purified1,4-diacetylpiperazine-2,5-dione.

The advantages and technical effects of the present invention are: inview of drug polymorphism, dominant drug crystalline form is extremelyimportant for the stability of drug quality. No polymorphs of(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione has been reportedso far. A thorough study on polyrnorphs of said compound has beenconducted in the present invention, and the inventors found that theδ-crystalline form of(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis a monohydrate, which is very stable in stress testing when exposed tohigh temperature, high humidity and illumination, and in acceleratedstability testing, while the α-crystalline form of said compound iseasily transformed into the δ-crystalline form under high humiditycondition, and the β, γ and ϵ-crystalline forms are not stable enoughunder high temperature condition which results in the loss ofcrystallization solvent, and partial crystalline forms may also betransformed into the δ- crystalline form under high humidity condition.Meanwhile, all β, γand ϵ-crystalline forms of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionecontain crystallization solvent, which exceeds the requirement onresidual solvent content in an API and therefore are not suitable fordrug development. Based on the aforementioned consideration andexperimental verification, said δ-crystalline form has excellentstability and safety.

Plinabulin has been in Phase III clinical study, and as its formulationis an injection, the stability of API is particularly important. Thepresent invention studies the polymorphs of plinabulin, and in light ofdrug stability of said polymorphs, the present invention determines thatthe d-crystalline form of (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl- 1H-imidazol-4-yl)methylene]piperazine-2,5-dione is a monohydrate, whereinsaid crystalline form is easily prepared with high stability. Whilea-crystalline form has certain hygroscopicity and is easily transformedinto a d-crystalline form under high humidity condition, b- and c-crystalline forms contain organic solvent which results in high residualsolvent and therefore are not suitable for drug development. Thed-crystalline form as a monohydrate is not easy to lose its watermolecule, and has excellent molecular stability during preparation andstorage processes. its quality and pharmaceutical efficacy during drugdevelopment are not altered. Therefore, the d-crystalline form is themost beneficial form of the compound disclosed herein.

The benzylidene group in the structure of both(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione and (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione is a double bond, whichis highly prone to cis-trans isomerization under illumination conditionto produce isomer impurities, posing certain safety risk for drugdevelopment. The present invention provides methods of preparing andpurifying monohydrates of both (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione and(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewith high purities, with the merits of being easy to operate, high yieldand high purity. Isomer impurities in the product can be controlled tobe less than 0.1%, and the aforementioned monohydrates as solid APIs canremain stable under high temperature, high humidity and illuminationconditions in stress testing, without isomerization or change in thecontent of impurities.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an X-ray powder diffraction pattern of the α-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneof the present invention.

FIG. 2 shows an X-ray powder diffraction pattern of the -crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneof the present invention.

FIG. 3 shows an analytical structure diagram of X-ray single crystaldiffraction of the β-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-y1)deuteromethylene]piperazine-2,5-dione of the present invention.

FIG. 4 shows an X-ray powder diffraction pattern of the y-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneof the present invention.

FIG. 5 shows an analytical structure diagram of X-ray single crystaldiffraction of the γ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneof the present invention.

FIG. 6 shows an X-ray powder diffraction pattern of the δ-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-deuteromethylene]piperazine-2,5-dioneof the present invention.

FIG. 7 shows an analytical structure diagram of X-ray single crystaldiffraction of the δ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione of the presentinvention, wherein a is the crystalline analytical structure obtained bymethod one, and b is the crystalline analytical structure obtained bymethod two.

FIG. 8 shows a thermogravimetric analysis diagram of the δ-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneof the present invention.

FIG. 9 shows an analytical structure diagram of X-ray single crystaldiffraction of the ϵ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneof the present invention.

FIG. 10 shows an X-ray powder diffraction pattern of the a-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneof the present invention.

FIG. 11 shows an X-ray powder diffraction pattern of the b-crystallineform of (3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneof the present invention.

FIG. 12 shows an analytical structure diagram of X-ray single crystaldiffraction of the b-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-H-imidazol-4-yl)methylene]piperazine-2,5-dioneof the present invention.

FIG. 13 shows an X-ray powder diffraction pattern of the c-crystallineform of(3Z,6Z)-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-l)methylene]piperazine-2,5-dioneof the present invention.

FIG. 14 shows an analytical structure diagram of X-ray single crystaldiffraction of the c-crystalline form of (3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneof the present invention.

FIG. 15 shows an X-ray powder diffraction pattern of the d-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneof the present invention.

FIG. 16 shows a thermogravimetric analysis diagram of the d-crystallineform of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione of the present invention.

FIG. 17 shows an analytical structure diagram of X-ray single crystaldiffraction of the d-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneof the present invention.

FIG. 18 shows a HPLC chromatogram of the monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionewith high purity of the present invention at a wavelength of 254 nm;

FIG. 19 shows a thermogravimetric analysis diagram of the monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionewith high purity of the present invention;

FIG. 20 shows a HPLC chrotnatogram of the monohydrate of(3Z6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewith high purity of the present invention at a wavelength of 254 nm;

FIG. 21 shows a thermogravimetric analysis diagram of the monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewith high purity of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

The technical solutions of the present invention are further describedin detail below with reference to the accompanying drawings and specificembodiments.

Example 1

Preparation of(3Z,6Z)-3-benzylidene-6-[(5-Cert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

The specific preparation process comprises the following steps:

1) Preparation of 5-(tert-butyl)oxazole-4-ethyl formate

90 g (796 mmol) of ethyl isocyanoacetate was added to 1000 mL oftetrahydrofuran, followed by slow dropwise addition of 145 g (955 mmol)of DBU and dropwise addition of 178 g (955 mmol) of trimethylaceticanhydride. The obtained was stirred at room temperature for 48 hours,after the reaction is completed, the reaction solution was concentratedunder reduced pressure. The concentrate was extracted, 1500 mL ofdichloromethane was added, followed by successive washing with 800 mL of10% sodium carbonate, 800 mL of 10% citric acid, 800 nit of saturatedbrine. The aqueous phase was back-extracted twice with 1000 mL ofdichloromethane. The organic phases were combined, dried over anhydroussodium sulfate for half an hour, suction filtered, concentrated underreduced pressure, and filtered through a silica gel (200-300 mesh)column(EA:PE=1:10, 1:8, 1:5) to give 177 g of said5-(tert-butyl)oxazole-4-ethyl formate as a yellow liquid.

2) Preparation of 5-(tert-butyl)-1H-imidazole-4-ethyl formate

157 g (796 mmol) of 5-(tert-butyl)-1H-oxazole-4-ethyl formate was addedto 717 g (15.914 mol) of formamide, let stand in an oil bath at 180° C.,stirred under reflux for 30 hours, cooled to room temperature, extractedand added with 800 mL of 10% sodium carbonate. 500 mL of petroleum etherwas added to extract and the organic phase was discarded. Then extractedthree times with ethyl acetate (1000 mL*3). The organic phases werecombined and washed twice with saturated brine (800 mL*2). The aqueousphase was back-extracted twice with ethyl acetate (500 mL*2). Theorganic phases were combined and dried over anhydrous sodium sulfate,suction filtered, concentrated under reduced pressure, pulpified,stirred after addition of 1000 mL of water and then suction filtered.The filter cake was washed with water and dried in vacuo at 50° C. togive 71 g of 5-(tert-butyl)-1H-imidazole-4-ethyl formate as a earthysolidwith a yield of 45%.

3) Preparation of 5-(tert-butyl)-1H-imidazole-4-methanol 40 g (1054mmol) of lithium aluminum hydride was added to 300 mit of driedtetrahydrofuran in a cold trap at −10° C., 70 g (357 mmol) of5-(tert-butyl)-1H-imidazole-4-ethyl formate in 200 mL of tetrahydrofuranwas slowly added dropwise to the turbid lithium aluminum hydridesolution. The mixture was moved to room temperature and stirred for 3hours before quenched by ice-water, the reaction mixture was addeddropwise to an appropriate amount of ice in a measuring cup. Theresulting mixture was suction filtered, and the filter cake wassuccessively washed twice with water (1000 mL*2), twice withtetrahydrofuran (500 mL*2), twice with anhydrous ethanol (500 mL* 2),followed by concentration under reduced pressure, dehydration once by95% ethanol and twice by anhydrous ethanol to give 51 g of said5-(tert-butyl)-1H-imidazole-4-methanol as a pale-yellow solid with ayield of 93%.

4) Preparation of 5-(tert-butyl)-1H-imidazole-4-formaldehyde 50 g (324mmol) of 5-(tert-butyl)-1H-imidazole-4-methanol was added to 500 mL ofdichloromethane, followed by addition of 282 g (3242 mmol) of manganesedioxide. The reaction was stirred at room temperature for 24 hours. Themixture was suction filtered and added with diatomite. The filter cakewas washed three times with anhydrous ethanol (500 mL*3) andconcentrated under reduced pressure to give 43 g of said5-(tert-butyl)-1H-imidazole-4-formaldehyde as a pale-yellow solid with ayield of 87%.

5) Preparation of 5-(tert-butyl)-1H-imidazole-4-deuteromethanol 17 g(112 mmol) of 5-(tert-butyl)-1H-imidazole-4-formaldehyde was dissolvedin 110 mL of ethanol at −10° C., followed by batchwise addition of 14 g(336 mmol) of sodium borodeuteride. The reaction was carried out for 20hours, quenched by 200 mL of saturated ammonium chloride andconcentrated under reduced pressure. The resulting concentrate wasextracted with 500 mL of saturated potassium carbonate and ethylacetate. The organic phases were combined, dried, concentrated withreduced pressure and pulpified with a 50:1 mixture of petroleum etherand ethyl acetate to give 13 g of said5-(tert-butyl)-1H-imidazole-4-deuteromethanol as a white solid with ayield of 74%.

6) Preparation of 5-(tert-butyl)-1H-imidazole-4-deuteroformaldehyde

12.90 g (82.57 mmol) of 5-(tert-butyl)-1H-imidazole-4-deuteromethanolwas added to 150 mL of dichloromethane, followed by addition of 71.79 g(825.74 mmol) of manganese dioxide. The reaction was stirred at 30° C.for 40 hours. The mixture was filtered by suction, washed with 1000 mLof ethyl acetate and concentrated under reduced pressure to give 10.00 gof said 5-(tert-butyl)-1H-imidazole-4-deuteroformaldehyde with a yieldof 79%.

7) Preparation of N,N-dia.cetylpiperazine-2,5-dione

50 g (438 mmol) of glycine anhydride was added into 179 g (1753 mmol) ofacetic anhydride. The mixture was placed in an oil bath at 155° C. andstirred under reflux for 30 hours, concentrated under reduced pressure,dissolved in dichloromethane and filtered through diatomite and silicagel. The resulting filter cake was rinsed with dichloromethane,concentrated under reduced pressure and recrystallized in ethyl acetateat 70° C. to give 74 g of said N,N-diacetylpiperazine-2,5-dione as abrown solid with a yield of 85%.

8) Preparation of(Z)-1-acetyl-3-[(5-(tert-butyl)-1-H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

10.00 g (65.27 mmol) of 5-(tert-butyl)-1H-imidazol-4-deuteroformaldehydewas added to 50 mL of DMF, followed by addition of 25.88 g (130.59 mmol)of N,N-diacetylpiperazine-2,5-dione. The resulting solution wasrepeatedly exhausted under nitrogen protection for three times, 31.91 g(97.94 mmol) of cesium carbonate was added and the mixture wasrepeatedly exhausted under nitrogen protection for three times. Thereaction was stirred at room temperature for 20 hours in the absence oflight, the reaction solution was then poured into ice-water (400 mL) andfiltered by suction. The resulting filter cake was successively washedwith water (200 mL*2) and an 8:1 mixture of petroleum ether and ethylacetate (200 mL), then ultrasonically dispersed in ethanol anddichloromethane. The insolubles was filtered off and the resultingfiltrate was concentrated under reduced pressure, dehydrated byanhydrous ethanol and pulpified with ethyl acetate (250 mL) to give 8.96g of said(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneas a brown-yellow solid with a yield of 47.11%.

9) Preparation of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deutromethylene]piperazine-2,5-dione

8.84 g (30.33 mmol) of (Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione was added to 25 mL of DMF,followed by addition of 4.83 g (45.51 mmol) of benzaldehyde. Theresulting solution was repeatedly exhausted under nitrogen protectionfor three times. 14.82 g (45.49 mmol) of cesium carbonate was added andthe mixture was repeatedly exhausted under nitrogen protection for threetimes. The temperature was programmed to reach 50° C. and the reactionwas stirred for 24 hours. The reaction solution was poured intoice-water (300 mL) and filtered by suction. The resulting filter cakewas successively washed with 200 mL*2 water and an 8:1 mixture ofpetroleum ether and ethyl acetate (200 mL), then ultrasonicallydispersed in ethanol (50 mL) and ethyl acetate (160 mL). The insolubleswas filtered off and the resulting filtrate was concentrated underreduced pressure, dehydrated by anhydrous ethanol, ultrasonicallydispersed in 150 mL of ethyl acetate, let stand at −18° C. overnight andfiltered by suction. The resulting filter cake was washed with icy ethylacetate (50 mL) to give 6.66 g of said(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazole-4-yl)deuteromethylene]piperazine-2,5-dione as a yellow-green solid with a yield of 65.09%. ¹H NMR (500MHz, dmso) δ12.30 (s, 1H), δ12.22 (s, 1H), 10.00 (brs, 1H), 7.82 (d,J=12.7 Hz, 1H), 7.51 (d, J=7.6 Hz, 2H), 7.40 (t, J=7.7 Hz, 2H), 7.30 (t,J=7.4 Hz, 1H), 6.73 (s, 1H), 1.37 (s, 9H).

Example 2

Purification process of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneand preparation of its αcrystalline form

6.66 g of above obtained crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionewas placed in a brown bottle, 400 mL of isopropanol was added underheating till said crude product was completely dissolved. 160 mL ofwater was added resulting in no crystalline precipitation. The resultingsolution was stirred and cooled to precipitate at room temperature andfiltered by suction. The resulting filter cake was washed with a 1:1mixture of isopropanol and water, pulpified with 100 mL of ethyl acetatefor 10 hours and filtered. The resulting filter cake was washed withethyl acetate and dried to give 5.323 g of a yellow powder solid with ayield of 80.0%. The crystalline form of the obtained solid wasα-crystalline form and its main X-ray powder diffraction characteristicpeaks at 2θ diffraction angle are shown in Table 1, and the specificdiffraction pattern is shown in FIG. 1.

Table 1 shows data on powder XRD analysis of the α, β, γ, δ-crystallineforms of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneof the present invention.

TABLE 1 Powder XRD characteristic peaks of the α-, β-, γ-, δ-crystallineforms α-crystalline β-crystalline γ-crystalline δ-crystalline Contenttested from form form from Main XRD 8.415° 7.629° 8.075° 8.075° powder11.512° 8.052° 12.986° 9.145° diffraction 12.271° 8.958° 16.217° 12.988°characteristic 13.126° 12.967° 19.709° 14.740° absorption 13.618°15.327° 24.441° 16.20.1° peaks 14.824° 16.195° 17.545° 16.011° 16.606°18.367° 16.282° 17.410° 19.084° 17.087° 23.194° 19.724° 17.278° 23.760°22.781° 17.608° 24.129° 23.710° 18.134° 24.419° 24.422° 18.408° 25.256°25.279° 19.461° 26.465° 26.485° 19.735° 29.213° 27.867° 20.745° 29.234°21.350° 22.344° 23.198° 24.874° 25.168° 26.997° 27.621° 28.479°

Example 3

Preparation of a β-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving

(3Z,6Z-benzylidene-6-[(5tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione(200 mg, 0.59 mmol) in a mixed solution of 20 mL of methanol and 0.1 mLof water at 70° C., filtering into a crystallizing dish with its openingcovered with a piece of in plastic wrap membrane in which holes weremade by a capillary having an outer diameter of 0.5 mm, letting stand tovolatilize in the absence of light at 25° C. to precipitate aβ-crystalline form of crystal 72 hours later, filtering and drying togive 142 mg of a cubic solid with a yield of 71%. The melting point ofthe obtained β-crystalline form is 263.6° C. to 264.4° C. The obtainedβ-crystalline form was examined by X-ray powder diffraction test, andits characteristic peaks at 2θ diffraction angle are shown in Table 1.The specific X-ray powder diffraction pattern is shown in FIG. 2.

The β-crystalline form prepared above is determined as a single crystalin which one molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione is combined with one molecule of methanol. X-ray singlecrystal diffraction test is carried out on a Bruker X-ray single crystaldiffractometer using Cu-Kα radiation (λ=1.54178 Å) at the temperature of293.2K, with compound size set to be 0.45 mm×0.43 mm×0.36 mm and datacollected at θ angle of 4.05 to 66.40 degrees. The test results ofcrystallographic parameters are shown in Table 2 below, and theanalytical structure is shown in FIG. 3.

TABLE 2 Crystallographic parameters Parameter Test results Empricalformula C₂₀H₂₃DN₄O₃ Molecular weight 369.44 Crystal system monoclinicSpace group P2(1)/n Unit cell dimentions a = 14.0192(7) Å α = 90 deg. b= 17.0390(8) Å β = 103.564(2) deg. c = 16.0709(11) Å γ = 90 deg. Volume3891.5(4) Å³ Z, calculated density 8, 1.261 Mg/m³ Absorption coefficient0.703 mm⁻¹ Number of electrons in a unit cell 1568 Crystallite size 0.45× 0.43 × 0.36 mm Limiting indices −161 ≤ h ≤ 17, −20 ≤ k ≤ 20, −18 ≤ l ≤19 Collected/unique diffraction data 13581/6839 [R(int) = 0.0411]Completeness to θ = 66.40 100.0% Absorption correction parametersSemi-empirical from equivalents Max. and min transmission 0.7860 and0.7428 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 6839/0/496 Goodness-of-fit on F² 1.056 FinalR indices R1 = 0.0576, wR2 = 0.1534 R indices (all data) R1 = 0.0904,wR2 = 0.1765 Light absorption coefficient 0.00105(12) Largest diff. peakand valley 0.223 and −0.188 e. Å⁻³

Example 4

Preparation of a γ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione(100 mg, 0.30 mmol) in a mixed solvent of 13 mL of methanol and 0.52 mLof water at 30° C., filtering into a crystallizing dish and inoculatingwith seed crystals, covering the opening of crystallizing dish with apiece of plastic wrap membrane in which holes were made by a capillarywith an outer diameter of 0.5 mm, letting stand to volatilize at 18° C.in the absence of light to precipitate a γ-crystalline form of crystal72 hours later, filtering and drying to give 38 mg of a diamond-shapesolid with a yield of 38%. The melting point of the obtainedγ-crystalline form is 263.6° C. to 265.1° C. The obtained γ-crystallineform was analyzed by X-ray powder diffraction test, and itscharacteristic peaks at 2θ diffraction angle are shown in Table 1. Thespecific X-ray powder diffraction pattern is shown in FIG. 4.

The γ-crystalline form is determined as a single crystal in which onemolecule of(3Z,6Z)-3-benzylidene-6-[(5-tertbutyl-1H-imidazol-4-yl)deuteromethylene]piperidine-2,5-dione is combined with two molecules of methanol. X-ray single crystaldiffraction test is carried out on a Bruker X-ray single crystaldiffractometer equipped with Cu-Kα radiation (γ=1.54178 Å) at thetemperature of 293K., with compound size set to be 0.50 mm×0.40 mm×0.18mm and data collected at θ angle of 3.79 to 66.38 degrees. The testresults of crystallographic parameters are shown in Table 3 below, andthe analytical structure is shown in FIG. 5.

TABLE 3 Crystallographic parameters Parameter Test results Empricalformula C₂₁H₂₇DN₄O₄ Molecular weight 401.48 Crystal system triclinicSpace group P-1 Unit cell dimensions a = 9.4485(9) Å α = 101.769(2) deg.b = 0.1149(10) Å β = 96.7290(10) deg. c = 12.1258(13) Å γ = 105.604(2)deg. Volume 1074.35(19) Å³ Z, calculated density 2, 1.241 Mg/m³Absorption coefficient 0.701 mm⁻¹ Number of electrons in a unit cell 428Crystallite size 0.50 × 0.40 × 0.18 mm Limiting indices −11 ≤ h ≤ 11, −7≤ k ≤ 12, −14 ≤ l ≤ 14 Collected/unique diffraction data 6293/3765[R(int) = 0.0289] Completeness to θ = 66.40 99.9% Absorption correctionparameters Semi-empirical from equivalents Max. and min transmission0.8828 and 0.7179 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 3765/0/267 Goodness-of-fit on F² 1.064 FinalR indices R1 = 0.0570, wR2 = 0.1555 R indices (all data) R1 = 0.0815,wR2 = 0.1789 Light absorption coefficient 0.0076(10) Largest diff. peakand valley 0.492 and −0.239 e. Å⁻³

Example 5

Preparation of a δ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving said(3Z,6Z)-3-benzylidene-6-[(tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionesolid (1.00 g, 2.96 mmol) in 60 mL of isopropanol at 80° C., filteringupon heating, letting the filtrate stand at 80° C., adding 12 ml ofwater dropwise, stirring the lo clear solution to precipitate at roomtemperature for 6 hours, filtering and drying to give 0.95 g of a yellowpowder crystalline solid with a yield of 90.41%. The obtainedδ-crystalline form was analyzed by X-ray powder diffraction testexhibiting characteristic absorption peaks at 2θ diffraction angle of8.075°, 9.145°, 12.988°, 14.740°, 16.201°, 17.545°, 18.367°, 19.084°,19.724°, 22.781°, 23.710°, 24.422°, 25.279°, 26.485°, 27.867°, 29.234°,the 2θ diffraction angle error is ±0.2°, see FIG. 6.

The obtained δ-crystalline form is a monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione,containing 5.314% water as determined by a Karl Fischer moisture meter.FIG. 8 shows the data support from a thermogravimetric analysis diagram,wherein the element analysis is shown in Table 4 below:

TABLE 4 Data on element analysis of the δ-crystalline form DeterminationTheratical Sample value (%) value (%) δ-crystalline form of C: 64.19 C:64.21 (3Z,6Z)-3-benzylidene-6- N: 15.78 N: 15.76[(5-tert-butyl-1H-imidazol-4-yl) H: 6.24 H: 6.27deuteromethylene]piperazine-2,5-dione

Example 6

Preparation of a δ-crystalline single crystalof(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

Method One

The specific preparation process comprises the following steps: weighingand dissolving

(3Z,6Z)-3-benzylidene-6[(tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione(100 mg, 0.30 mmol) in a mixed solvent of 13 mL of methanol and 0.52 mLof water at 30° C., filtering into a crystallizing dish and inoculatingwith seed crystals, covering the opening of crystallizing dish with apiece of plastic wrap membrane in which holes were made using acapillary with an outer diameter of 0.5 mm, letting stand to volatilizeat 13° C. in the absence of light to precipitate a δ-crystalline form ofcrystal 72 hours later, filtering and drying to give 34 mg of aneedle-like solid with a yield of 34%. The melting point of theobstained δ-crystalline form is 264.4° C. to 266.2° C., and itscharacteristic absorption peaks at 2θ diffraction angle analyzed byX-ray powder diffraction test are consistent with those of FIG. 6.

The δ-crystalline form prepared above is determined as a single crystalin which one molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis combined with one molecule of water. X-ray single crystal diffractiontest is carried out on a Bruker X-ray single crystal diffractometerequipped with Cu-Kα radiation (γ=1.54178 Å) at the temperature of 293K,with compound size set to be 0.42mm×0.41mm×0.40 mm and data collected atθ angle of 4.05 to 66.40 degrees. The test results of crystallographicparameters are shown in Table 5-1 below, and the analytical structure isshown as structure a in FIG. 7.

TABLE 5-1 Crystallographic parameters Parameter Test results Empricalformula C₁₉H₂₁DN₄O₃ Molecular weight 355.41 Crystal system monoclinicSpace group P2(1)/c Unit cell dimensions a = 9.7051(5) Å α = 90 deg. b =8.5247(5) Å β = 97.6520(10) deg. c = 22.0068(12) Å γ = 90 deg. Volume1804.48(17) Å³ Z, calculated density 4, 1.308 Mg/m³ Absorptioncoefficient 0.737 mm⁻¹ Number of electrons in a unit cell 752Crystallite size 0.42 × 0.41 × 0.40 mm Limiting indices −6 ≤ h ≤ 11, −9≤ k ≤ 10, −26 ≤ l ≤ 26 Collected/unique diffraction data 6052/3171[R(int) = 0.0326] Completeness to θ = 66.40 100.0% Absorption correctionparameters Semi-empirical from equivalents Max. and min transmission0.7569 and 0.7470 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 3171/0/240 Goodness-of-fit on F² 1.055 FinalR indices R1 = 0.0483, wR2 = 0.1269 R indices (all data) R1 = 0.0659,wR2 = 0.1408 Light absorption coefficient 0.0210(10) Largest diff. peakand valley 0.237 and −0.194 e. Å⁻³

Method Two

The specific preparation process comprises the following steps: weighingand dissolving

(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione(100 mg, 0.30 mmol) in a mixed solvent of 15 rriL of anhydrous ethanoland 1 mL of water at 65° C., filtering into a crystallizing dish withits opening covered with a piece of plastic wrap membrane in which holeswere made by a capillary with an outer diameter of 0.5 mm, letting standto volatilize at 26° C. in the absence of light to precipitate aδ-crystalline form of crystal 72 hours later, filtering and drying togive 51 mg of a long columnar solid with a yield of 49%. The meltingpoint of the δ-crystalline form obtained is 264.9° C. to 266.1° C., andits characteristic absorption peaks at 2θ diffraction angles analyzed byX-ray powder diffraction test are consistent with those of FIG. 6.

The δ-crystalline form prepared above is determined as a single crystalin which one molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis combined with one molecule of water. X-ray single crystal diffractiontest is carried out on a Bruker X-ray single crystal diffractometerusing Cu-Kα radiation(λ=1.54178 Å) at the temperature of 293K, withcompound size set to be 0.45 mm×0.43 mmx0.32mm and data collected at θangle of 4.05 to 66.20 degrees. The test results of crystallographicparameters are shown in Table 5-2 below, and the analytical structure isshown as structure b in FIG. 7.

TABLE 5-2 CrystalIographic parameters Parameter Test results Empricalformula C₁₉H₂₁DN₄O₃ Molecular weight 355.41 Crystal system monoclinicSpace group P2(1)/c Unit cell dimensions a = 9.7077(5) Å α = 90 deg. b =8.5222(5) Å β = 97.652(2) deg. c = 22.0126(12) Å γ = 90 deg. Volume1804.91(17) Å³ Z, calculated density 4, 1.308 Mg/m³ Absorptioncoefficient 0.737 mm⁻¹ Number of electrons in a unit cell 752Crystallite size 0.45 × 0.43 × 0.32 mm Limiting indices −11 ≤ h ≤ 11, −7≤ k ≤ 10, −24 ≤ l ≤ 26 Collected/unique diffraction data 5797/3149[R(int) = 0.0335] Completeness to θ = 66.20 100.0% Absorption correctionparameters Semi-empirical from equivalents Max. and min transmission0.7983 and 0.7326 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 3149/0/240 Goodness-of-fit on F² 1.038 FinalR indices R1 = 0.0492, wR2 = 0.1320 R indices (all data) R1 = 0.0620,wR2 = 0.1425 Light absorption coefficient 0.0306(15) Largest diff. peakand valley 0.225 and −0.186 e. Å⁻³

Example 7

Hygroscopicity test of the δ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

The test was conducted according to the general rule 9103 of guidingprinciple for drug hygroscopicity test in Chinese Pharmacopoeia 2015edition Volume IV, and the results are shown in Table 6.

TABLE 6 Hygroscopicity test results Left for 24 h, Weight of Weight ofWeight of Sample + Sample + Weight Batch weighing weighing weighing gainno. bottle (mg) bottle (mg) bottle (mg) (mg) Hygroscopicity 2015040130117.46 3042.3.60 30423.80 0.20 none 20150402 32498.44 32801.1732801.41 0.24 none 20150403 29458.09 29765.76 29765.96 0.20 none

With bottle opened for 24 hours, weight gain percentage of theδ-crystalline form were less than 0.2%, indicating the δ-crystallineform is basicly not hygroscopic according to the definition onhygroscopicity and hygroscopic weight gain in the Chinese Pharmacopoeia.

Studies on other crystalline forms of (3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneindicate that the α-crystalline form has certain hygroscopicity and maypartially transform into δ-crystalline form under 60% humiditycondition. Meanwhile, the β and γ-crystalline forms have nohygroscopicity, but most of which may transform into δ-crystalline formafter being stirred in water for 3 hours.

Example 8

Stress testing and accelerated stress testing of the δ-crystalline formof(3Z,6Z-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

In accordance with the general rule 9001 of guiding. principles forstability tests of

APIs and preparations in the Chinese Pharmacopoeia 2015 edition VolumeIV, stress testing of the δ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionewas conducted under high temperature, high humidity and illuminationconditions, the sample batch number was 20150401, and the results areshown in Tables 7, 8 and 9 below.

TABLE 7 Data on high-temperature stress test of the δ-crystalline formItem High-temperature test/60° C. (days ) tested Limit requirement 0 510 Appearance Yellow crystalline Yellow powder Yellow powder Yellowpowder powder Relevant Impurity A ≤0.1% Impurity A: <LOQ Impurity A:<LOQ Impurity A: <LOQ substance Impurity D ≤0.1% (0.035%) (0.035%)(0.035%) (HPLC) Other single Impurity D: 0.06% Impurity D: 0.06%Impurity D: 0.06% impurity ≤0.1% Other single Other single Other singleTotal impurities ≤0.5% impurity: not impurity: not impurity: notdetected detected detected Total impurities: Total impurities: Totalimpurities: 0.07% 0.07% 0.07% Content 98.0%-102.0% 99.7% 99.4% 99.8%(HPLC) (calculated as an anhydride)

TABLE 8 Data on high-humidity stress test of the δ-crystalline form ItemHigh-humidity test/25° C., 90%RH (days) tested Limit requirement 0 5 10Appearance Yellow crystalline Yellow powder Yellow powder Yellow powderpowder Relevant Impurity A ≤0.1% Impurity A: <LOQ Impurity A: <LOQImpurity A: <LOQ substance Impurity D ≤0.1% (0.035%) (0.035%) (0.035%)(HPLC) Other single Impurity D: 0.06% Impurity D: 0.06% Impurity D:0.06% impurity ≤0.1% Other single Other single Other single Totalimpurities ≤0.5% impurity: not impurity: not impurity: not detecteddetected detected Total impurities: Total impurities: Total impurities:0.07% 0.07% 0.07% Content 98.0%-102.0% 99.7% 99.5% 99.6% (HPLC)(calculated as an anhydride)

TABLE 9 Data on illumination stress test of the δ-crystalline form ItemIllummatioin test/4500Lx ± 500Lx ( days ) tested Limit requirement 0 510 Appearance Yellow crystalline Yellow powder Yellow powder Yellowpowder powder Relevant Impurity A ≤0.1% Impurity A: <LOQ Impurity A:<LOQ Impurity A: <LOQ substance Impurity D ≤0.1% (0.035%) (0.035%)(0.035%) (HPLC) Other single Impurity D: 0.06% Impurity D: 0.05%Impurity D: 0.05% impurity ≤0.1% Other single Other single Other singleTotal impurities ≤0.5% impurity: not impurity: not impurity: notdetected detected detected Total impurities: Total impurities: Totalimpurities: 0.07% 0.05% 0.05% Content 98.0%-102.0% 99.7% 100.1% 99.8%(HPLC) (calculated as an anhydride)

Experiments show that the δ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis relatively stable when let stand in high-temperature (60° C.),high-humidity (25° C., 90% RH) or illumination (4500 Lx±500 Lx)condition for 10 days. The α-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneabsorbs moisture easily to transform into δ-crystalline form underhigh-humidity condition, and the β-, γ- and ϵ-crystalline forms are notstable enough under high-temperature condition causing the loss ofcrystallization solvents, and some of these crystalline forms maytransform into δ-crystalline form under high-humidity condition. As theβ-, γ- and ϵ-crystalline forms of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione contain crystallization solvent, exceeding thecorresponding limit requirement in APIs, therefore these threecrystalline forms are not suitable for further drug development. Basedon the aforementioned consideration and experimental verification, theδ-crystalline form shows excellent stability and safety.

Meanwhile, a 6-month accelerated stability test was conducted on theδ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2.5-dione.The sample batch number was 20150404. Factory packaging was simulated.

The test conditions were 40° C.±2° C./75%±5% RH. The results are shownin Table 10 below.

TABLE 10 Data on accelerated stability test of the δ-crystalline formItem Limit Observation period (months) tested requirement 0 1 2 3 6Apperance Yellow Yellow Yellow Yellow Yellow Yellow crystalline powderpowder powder powder powder powder Relevant Impurity <LOQ <LOQ <LOQ <LOQ<LOQ substance A <0.1% (0.035%) (0.035%) (0.035%) (0.035%) (0.0350%)(HPLC) Impurity  0.06%  0.06%  0.06%  0.06%  0.06% D ≤0.1% Not detectedNot detected Not detected Not detected Not detected Other single  0.07% 0.08%  0.08%  0.08%  0.08% impurity ≤0.1% Total impurities ≤0.5%Content 98.0%-102.0%  99.5%  99.6% 100.2%  99.8%  99.7% (HPLC)(Calculated as an anhydride)

Results indicate that tlre δ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneshows excellent stability during the 6-month accelerated stress test,which provides a solid foundation for its pharmaceutical research.

Example 9

Preparation of an ϵ-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving

(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione(100 mg, 0.30 mmol) in a mixed solvent of 15 mL, of isopropanol and 0.30mL of purified water at 55° C., filtering into a crystallizing dish withits opening covered with a piece of plastic wrap membrane in which holeswere made using a capillary with is an outer diameter of 0.5 mm, lettingstand to volatilize at 15° C. in the absence of light to precipitate anc-crystalline form 48 hours later, filtering and drying to give 45 mg ofa diamond-shape solid with a yield of 45%. The melting point of obtainedϵ-crystalline form is 264.1° C. to 264.7° C.

The ϵ-crystalline form is determined as a single crystal in which onemolecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneis combined with one molecule of isopropanol. X-ray single crystaldiffraction test is carried out on a Bruker X-ray single crystaldiffractometer using Cu-Kα radiation (λ=1.54178 Å) at the temperature of293K, with compound size set to be 0.42 mm×0.28 mm×0.12mm and datacollected at θ angle of 3.82 to 66.05 degrees. The results ofcrystallographic parameters test are shown in Table 11 below, and theanalytical structure is shown in FIG. 9.

TABLE 11 Crystallographic parameters Parameter Test results Empricalformula C₂₂H₂₇DN₄O₄ Molecular weight 397.49 Crystal system monoclinicSpace group P2(1)/c Unit cell dimensions a = 11.7836(10) Å α = 90 deg. b= 8.3648(9) Å β = 101.181(9) deg. c = 22 778(2) Å γ = 90 deg. Volume2202.6(4) Å³ Z, calculated density 4, 1.199 Mg/m³ Absorption coefficient0.654 mm⁻¹ Number of electrons in a unit cell 752 Crystallite size 0.42× 0.28 × 0.12 mm Limiting indices −13 <= h <= 10, −9 <= k <= 9, −26 <= l<= 26 Collected/unique diffraction data 7493/3832 [R(int) = 0.0559]Completeness to θ = 66.40 99.9% Absorption correction parametersSemi-empirical from equivalents Max. and min transmission 0.9257 and0.7708 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 3832/0/295 Goodness-of-fit on F² 1.081 FinalR indices R1 = 0.0646, wR2 = 0.1372 R indices (all data) R1 = 0.1223,wR2 = 0.1700 Light absorption coefficient 0.0088(5) Largest diff. peakand valley 0.245 and −0.235 e. Å⁻³

Example 10

Preparation of an a-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

The specific preparation process includes the following steps:

1) Preparation of 5-(tert-butyl)oxazole-4-ethyl formate

90 g (796 mmol) of ethyl isocyanoacetate was added to 1000 mL oftetrahydrofuran, followed by slow dropwise addition of 145 g (955 mmol)of DBU and dropwise addition of 178 g (955 mmol) of trimethylaceticanhydride. The reaction was stirred at room temperature for 48 hours.After the reaction was complete, the mixture was concentrated underreduced pressure. Extracted, an appropriate amount of 1500 mL ofdichloromethane was added, followed by successive washing with 800 mL of10% sodium carbonate, 800 mL of 10% citric acid and 800 mL of saturatedbrine. The aqueous phase was back-extracted twice with 1000 mL ofdichloromethane. The organic phases were combined, dried over anhydroussodium sulfate for half an hour, filtered by suction, concentrated underreduced pressure and filtered through silica gel (200-300 mesh) column(EA: PE=1:10, 1:8, 1:5) to give 177 g of said5-(tert-butyl)oxazole-4-ethyl formate as a yellow liquid with a yield of113%.

2) Preparation of 5-(tert-butyl)-11-1-imidazole-4-ethyl formate

157 g (796 mmol) of 5-(tert-butyl)-1H-oxazole-4-ethyl formate was addedto 717 g (15.914 mmol) of formamide. The mixture was let stand in an oilbath at 180° C. and stirred under reflux for 30 hours, cooled to roomtemperature, extracted and added with 800 ml of 10% sodium carbonate.500 mL of petroleum ether was added to extract and the organic phase wasdiscarded. The obtained was then extracted three times with ethylacetate (1000 mL*3). The organic phases were combined, washed twice withsaturated brine (800 mL*2) and the aqueous phase was back-extractedtwice with ethyl acetate (500 mL*2). The organic phases were combined,dried over anhydrous sodium sulfate, filtered by suction, concentratedunder reduced pressure, pulpified, added with 1000 mL of water, stirredand filtered by suction. The filter cake was washed with water and driedin vacuo at 50° C. to give 71 g of said5-(tert-butyl)-1H-imidazole-4-ethyl formate as a earthy-yellow solidwith a yield of 45%.

3) Preparation of 5-(tert-butyl)-1H-imidazole-4-methanol

40 g (1054 mmol) of lithium aluminum hydride was added to 300 mL ofdried tetrahydrofuran in a cold trap at −10° C. 70 g (357 mmol) of5-(tert-butyl)-1H-imidazole-4-ethyl formate in 200 mL of tetrahydrofuranwas slowly added dropwise to the turbid lithium aluminum hydridesolution. The reaction was stirred at room temperature for 3 hours.Quenched by ice-water, the reaction solution was added dropwise into ameasuring cup with an appropriate amount of ice. The mixture wasfiltered by suction, and the resulting filter cake was successivelywashed twice with water (1000 mL*2), twice with tetrahydrofuran (500mL*2), twice with anhydrous ethanol (500 mL*2), concentrated underreduced pressure, and dehydrated by anhydrous ethanol to give 51 g ofsaid 5-(tert-butyl)-1H-imidazole-4-methanol as a pale-yellow solid witha yield of 93%.

4) Preparation of 5-(tert-butyl)-1H-imidazole-4-carbaldehyde

50 g (324 mmol) of 5-(tert-butyl)-1H-imidazole-4-methanol was added to500 mL of dichloromethane, followed by addition of 282 g (3242 mmol) ofmanganese dioxide. The reaction was stirred at room temperature for 24hours. The mixture was filtered by suction and added with diatomite. Theresulting filter cake was washed three times with anhydrous ethanol (500mL*3) and concentrated under reduced pressure to give 43 g of said5-(tert-butyl)-1H-imidazole-4-carbaldehyde as a pale-yellow solid with ayield of 87%.

5) Preparation of N,N-diacetylpiperazine-2,5-dione

50 g (438 mmol) of glycine anhydride was added to 179 g (1753 mmol) ofacetic anhydride. The mixture was let stand in an oil bath at 155° C.,stirred under reflux for 30 hours, concentrated under reduced pressure,dissolved in dichloromethane and filtered through diatomite and silicagel. The filter cake was rinsed with dichloromethane, concentrated underreduced pressure, dissolved in ethyl acetate at 70° C. andrecrystallized to give 74 g of N,N-diacetylpiperazine-2,5-dione as abrown solid with a yield of 85%.

6) Preparation of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

1 g (6.5 mmol) of 5-(tert-butyl)-1H-imidazole-4-carbaldehyde was addedto 7 mL of DMF, followed by addition of 2.59 g (13 mmol) ofN,N-diacetylpipera.zine-2,5-dione. The resulting solution was repeatedlyexhausted under nitrogen for three times. 3.19 g (9.8 mmol) of cesiumcarbonate was added and the mixture was repeatedly exhausted undernitrogen for three times. The reaction was stirred at room temperaturefor 20 hours in the absence of light. The reaction solution was pouredinto ice-water (100 mL) and filtered by suction. The resulting filtercake was successively washed with water (100 mL*2) and an 8:1 mixture ofpetroleum ether and ethyl acetate (90 mL), then ultrasonically dispersedin ethanol and dichloromethane. The insolubles was filtered off and theresulting filtrate was concentrated under reduced pressure, dehydratedby anhydrous ethanol and pulpified with ethyl acetate (50 mL) to give0.89 g of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneas a brown-yellow solid with a yield of 46.9%.

7) Preparation of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione0.85 g (2.9 mmol) of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewas added to DMF, followed by addition of 0.56 g (5.25 mmol) ofbenzaldehyde. The resulting solution was repeatedly exhausted undernitrogen for three times. 0.95 g (2.9 mmol) of cesium carbonate wasadded and the mixture was repeatedly exhausted under nitrogen for threetimes. The temperature was programmed to reach 80° C. and the reactionwas stirred for 24 hours in the absence of light. The reaction solutionwas poured into ice-water (100 mL) and filtered by suction, Theresulting filter cake was washed successively with water (100mL*2) andan 8:1 mixture of petroleum ether and ethyl acetate (90 mL), thenultrasonically dispersed in ethanol (30 mL) and ethyl acetate (100 mL).The insolubles was filtered off and the resulting filtrate wasconcentrated under reduced pressure, dehydrated by anhydrous ethanol,dispersed with 50 mL of ethyl acetate, let stand at −30° C. overnightand filtered by suction. The resulting filter cake was washed with icyethyl acetate (5 mL) to give 0.59 g of an a-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionee as a yellow powder solid with a yield of 60.1%.

¹H NMR (400 MHz, DMSO-d6)δ 12.31 (s, 1H), 12.22 (s, 1H), 10.00 (S, 1H),7.84 (s, 1H), 7.52 (d, J=8 Hz, 2H), 7.39 (t, J=8 Hz, 2H), 7.32 (t, J=8Hz, 1H), 6.86 (s, 1H), 6.73 (s, 1H), 1.37 (s, 9H); MS (ESI) m/z 338.1715(M+H)⁺ (calcd for C₁₉H₂₁N₄O₂ 338.1722). As shown in FIG. 10, thea-crystalline form has an X-ray powder diffraction pattern comprisingcharacteristic peaks at 2θ angle of 8.446°, 11.546°, 12.285°, 13.161°,14.855°, 16.043°, 16.647°, 17.136°, 17.639°, 18.130°, 18.459′, 19.473°,19.804°, 20.742°, 21.343°, 22.338°, 23.235°, 24.868°, 25.145°, 27.596°,

Example 11

Preparation of a b-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving said(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione(200 mg, 0.59 mmol) in a mixed solution of 20 mL of methanol and 0.1 mLof water at 70° C., filtering into a crystallization dish with itsopening covered with a piece of plastic wrap membrane in which holeswere made using a capillary with an outer diameter of 0.5 ram, lettingstand to volatilize at room temperature in the absence of light toprecipitate a b-crystalline form 72 hours later, filtering and drying togive 148 mg of a cubic solid with a yield of 74%. X-ray powderdiffraction analysis shows that the obtained b-crystalline form exhibitscharacteristic absorption peaks at 20 angle of 7.356°, 7.670°, 8.097°,9.069°, 12.032°, 12.500°, 13.063°, 15.383°, 16.241°, 16.668°, 17.468°,18.109°, 18.694°, 19.960°, 23.307°, 23.836°, 24.462°, 28.046°, 28.827°,30.226°, the 2θ diffraction angle error is ±0.2° . The specific X-raypowder diffraction pattern is shown in FIG, 11. The melting point ofobtained b-crystalline form is 264.0° C. to 2649° C.

The obtained b-crystalline form is determined as a single crystal inwhich one molecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneis combined with one molecule of methanol. X-ray single crystaldiffraction test is carried out on a Bruker X-ray single crystaldiffractometer using Cu-Kα radiation (λ=1.54178 Å) at the temperature of293K, with compound size set to be 0.45mm×0.43mm×0.32mm and datacollected at θ angle of 4.05 to 66.40 degrees. The 2.0 test results ofcrystallographic parameters are shown in Table 12 below, and theanalytical structure is shown in FIG. 12.

TABLE 12 Crysiallographic parameters Parameter Test results Empricalformula C₂₀H₂₄N₄O₃ Molecular weight 368.43 Crystal system monoclinicSpace group P2(1)/n Unit cell dimensions a = 14.6268(6) Å α = 90 deg. b= 17.0440(6) Å β = 103.575(2) deg. c = 16.0850(8) Å γ = 90 deg. Volume3898.0(3) Å³ Z, calculated density 8, 1.256 Mg/m³ Absorption coefficient0.701 mm⁻¹ Number of electrons in a unit cell 1568 Crystallite size 0.45× 0.43 × 0.32 mm Limiting indices −11 ≤ h ≤ 17, −20 ≤ k ≤ 19, −19 ≤ l ≤18 Collected/unique diffraction data 13871/6847 [R(int) = 0.0318]Completeness to θ = 66.40 100.0% Absorption correction parametersSemi-empirical from equivalents Max. and min transmission 0.8067 and0.7431 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 6847/0/498 Goodness-of-fit on F² 1.018 FinalR indices R1 = 0.0534, wR2 = 0.1362 R indices (all data) R1 = 0.0812,wR2 = 0.1541 Light absorption coefficient 0.00092(9) Largest diff. peakand valley 0.221 and −0.169 e. Å⁻³

Example 12

Preparation of a c-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving said

(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione(200 mg, 0.59 mmol) in a mixed solution of 20 mL of methanol and 0.8 mLof water at 68° C., filtering into a crystallization dish andinoculating with seed crystals, covering the opening of crystallizationdish with a piece of plastic wrap membrane in which holes were madeusing a capillary with an outer diameter of 0.5 mm, letting stand tovolatilize at room temperature in the absence of light to precipitate ac-crystalline form 72 hours later, filtering and drying to give 98 mg ofa needle-like solid with a yield of 49%. X-ray powder diffractionanalysis shows that the obtained c-crystalline form exhibitscharacteristic absorption peaks at 2θ angle of 7.918°, 9.168°, 9.905°,12.014°, 12.985°, 14.970°, 15.873°, 18.382°, 18.616°, 19.081°, 19.881°,22.862°, 23.367°, 23.719°, 24.073°, 25.203°, 26.447°, 27.771°, 37.787°,the 2θ diffraction angle error is ±0.2°. The X-ray powder diffractionpattern is shown in FIG. 13 and the melting point of obtainedc-crystalline form is 263.2-264.0° C.

The obtained c-crystalline form is a single crystal in which onemolecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneis combined with one molecule of methanol. X-ray single crystaldiffraction test is carried out on a Bruker X-ray single crystaldiffractometer using Cu-Kα radiation (λ=1.54178 Å) at the temperature of293K, with compound size set to be 0.45 mm×0.30 mm×0.23 mm and datacollected at θ angle of 4.05 to 66.40 degrees. The test results ofcrystallographic parameters are shown in Table 13 below, and theanalytical structure is shown in FIG. 14.

TABLE 13 Crystallographic parameter table Parameter Test resultsEmpirical formula C₂₀H₂₄N₄O₃ Molecular weight 368.43 Crystal systemmonoclinic Space group P2(1)/n Unit cell dimensions a = 14.6218(11) Å α= 90 deg. b = 17.0401(11) Å β = 103.604(2) deg. c = 16.0771(13) Å γ = 90deg. Volume 3893.3(5) Å³ Z, calculated density 8, 1.257 Mg/m³ Absorptioncoefficient 0.702 mm⁻¹ Number of electrons in a unit cell 1568Crystallite size 0.45 × 0.30 × 0.23 mm Limiting indices −17 ≤ h ≤ 13,−20 ≤ k ≤ 16, −18 ≤ l ≤ 19 Collected/unique diffraction data 14326/6843[R(mt) = 0.0459] Completeness to θ = 66.40 100.0% Absorption correctionparameters Semi-empirical from equivalents Max. and min transmission 08552 and 0.7429 Refinement method Full-matrix least-squares on F²Data/restraints/pararneters 6843/0/496 Goodness-of-fit on F² 1.017 FinalR indices R1 = 0.0562, wR2 = 0.1340 R indices (all data) R1 = 0.1071,wR2 = 0.1635 Light absorption coefficient 0.00126(11) Largest diff. peakand valley 0.164 and −0.161 e. Å⁻³

The solvent molecules binding to the hydrogen bond in the conformationof obtained b-(as shown in FIG. 11) and c-(as shown in FIG. 14)crystalline forms of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneare not limited to methanol, also include molecules that are prone tobinding to the hydrogen bond in a carbonyl group such as other alkanesaturated alcohols, unsaturated alcohols, alkane saturated amines orunsaturated amines.

Example 13

Preparation of a d-crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving said(3Z,6Z)3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionesolid (1.00 g, 2.96 mmol) in 60 mL of isopropanol at 80° C., filteringupon heating, letting the resulting filtrate stand at 80° C., adding 12mL of water dropwise, stirring the resulting clear solution at roomtemperature to precipitate for 6 hours, filtering and drying to give0.96 g of a yellow powdery crystalline solid with a yield of 91.61%.X-ray powder diffraction analysis shows that the obtained d-crystallineform exhibits characteristic absorption peaks at 2θ angle of 8.073°,9.146°, 13.005°, 14.740°, 16,184°, 17.544°, 18.382°, 19.082°, 19.707°,22.766°, 23.759°, 24.438°, 25.277°, 26.486°, 27.883°, 29.234°, the 2θdiffraction angle error is ±0.2°. The X-ray powder diffraction patternis shown in FIG. 15, and the melting point of obtained d-crystallineform is 264.5° C. to 266.3° C.

The obtained d-crystalline form is a monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione,containing 5.326% water as determined by a Karl Fischer moisture meter.FIG. 16 shows the supporting data provided by a therrnogravimetricanalysis.

Table 14 shows data on powder XRD analysis of the a-, b-, c- andd-crystalline forms of(3Z,6Z)-3-benzylidene-6-[(tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5dioneof the present invention.

TABLE 14 Data on powder XRD analysis of the a-, b-, c- and d-crystallineforms Content a-crystalline b-crystalline c-crystalline d-crystallinetested form form form form Main 8.446° 7.356° 7.918° 8.073°characteristic 11.546° 7.670° 9.168° 9.146° absorption 12.285° 8.097°9.905° 13.005° peaks of 13.161° 9.069° 12.014° 14.740° powder 14.855°12.032° 12.985° 16.184° XRD 16.043° 12.500° 14.970° 17.544° 16.647°13.063° 15.873° 18.382° 17.136° 15.383° 18.382° 19.082° 17.639° 16.241°18.616° 19.707° 18.130° 16.668° 19.081° 22.766° 18.459° 17.468° 19.881°23.759° 19.473° 18.109° 22.862° 24.438° 19.804° 18.694° 23.367° 25.277°20.742° 19.960° 23.719° 26.486° 21.343° 23.307° 24.073° 27.883° 22.338°23.836° 25.203° 29.234° 23.235° 24.462° 26.447° 24.868° 28.046° 27.771°25.145° 28.827° 37.787° 27.596° 30.226°

Example 14

Preparation of a d-crystalline single crystal of(3Z,6Z)-3-berizylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

The specific preparation process comprises the following steps: weighingand dissolving

(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione(100 mg, 0.30 mmol) in a mixed solvent of 15 mL of anhydrous ethanol and1 mL of water at 65° C., filtering into a crystallization dish with itsopening covered with a piece of plastic wrap membrane in which 16 holeswere made using a capillary with an outer diameter of 0.5 mm, lettingstand to volatilize at 25° C. in the absence of light to precipitate ad-crystalline form 72 hours later, filtering and drying to give 52 mg ofa long columnar solid with a yield of 49.5%. The melting point ofobtained d-crystalline form is 264.2° C. to 265.3° C.,

The d-crystalline form is determined as a single crystal in which onemolecule of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneis combined with one molecule of water. X-ray single crystal diffractiontest is carried out on a Bruker X-ray single crystal diffractometerusing Cu-Kα radiation (λ=1.54178 Å) at the temperature of 293K, withcompound size set to be 0.41 mm×0.40 mm×0.30 mm and data collected at θangle of 4.06 to 66.19 degrees. The test results of crystallographicparameters are shown in Table 15 below, and the analytical structure isshown in FIG. 17.

TABLE 15 Crystallographic parameters Parameter Test results Empiricalformula C₁₉H₂₂N₄O₃ Molecular weight 354.41 Crystal system monoclinicSpace group P2(1)/c Unit cell dimensions a = 9.7047(6) Å α = 90 deg. b =8.5230(7) Å β = 97.6060(10) deg. c = 21.9790(15) Å γ = 90 deg. Volume1802.0(2) Å³ Z, calculated density 4, 1.306 Mg/m³ Absorption coefficient0.738 mm⁻¹ Number of electrons in a unit cell 752 Crystallite size 0.41× 0.40 × 0.30 mm Limiting indices −8 ≤ h ≤ 11, −9 ≤ k ≤ 10, −26 ≤ l ≤ 25Collected/unique diffraction data 5932/3139 [R(int) = 0.0398]Completeness to θ = 66.19 99.9% Absorption correction parametersSemi-empirical from equivalents Max. and min transmission 0.8089 and0.7517 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 3139/0/239 Goodness-of-fit on F² 1.069 FinalR indices R1 = 0.0597, wR2 = 0.1506 R indices (all data) R1 = 0.0763,wR2 = 0.1626 Light absorption coefficient 0.0254(13) Largest diff. peakand valley 0.220 and −0.204 e. Å⁻³

Example 15

Preparation of a crude product of(3Z,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

The specific preparation process includes the following steps:

1) Preparation of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4yl)deuteromethylene]piperazine-2,5-dione

10.00 g (65.29 mmol) of 5-(tert-butyl)-1H-imidazol-4-deuteroformaldehydewas added to 50 mL of DMF, followed by addition of 25.88 g (130.59 mmol)of N,N-diacetylpiperazine-2,5-dione. The resulting solution wasrepeatedly exhausted under nitrogen for three times. 31.91 g (97.94mmol) of cesium carbonate was added and the mixture was repeatedlyexhausted under nitrogen for three times. The reaction was stirred atroom temperature for 20 hours in the absence of light. The reactionsolution was poured into ice-water (400 mL), filtered by suction, andthe resulting filter cake was successively washed with water (200 mL*2)and a 8:1 mixture of petroleum ether and ethyl acetate (200 mL), thenultrasonically dispersed in ethanol and dichloromethane. The insolubleswas filtered off and the resulting filtrate was concentrated underreduced pressure, dehydrated by anhydrous ethanol and then pulpifiedwith ethyl acetate (250 mL) to give 8.96 g of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dioneas a brown-yellow solid with a yield of 47.11%. 2) Preparation of acrude product of (3Z,6Z)-3-benzy 1 idene-6-[(5-tert-buty-1E1-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione

8.84 g (30.33 mmol) of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dione was added to 25 mL of DMF, followed by addition of 4.83 g(45.51 mmol) of benzaldehyde. The resulting solution was repeatedlyexhausted under nitrogen for three times. 14.82 g (45.49 mmol) of cesiumcarbonate was added and the mixture was repeatedly exhausted undernitrogen for three times. The temperature was programmed to reach 50° C.and the reaction was stirred for 24 hours. The reaction solution waspoured into ice-water (300 mL), filtered by suction, and the resultingfilter cake was washed successively with water (200 mL*2) and an 8:1mixture of petroleum ether and ethyl acetate (200 mL), thenultrasonically dispersed in ethanol (50 mL) and ethyl acetate (160 mL).The insolubles was filtered off and the resulting filtrate wasconcentrated under reduced pressure, dehydrated by anhydrous ethanol,ultrasonically dispersed in 150 mL of ethyl acetate, let stand at −30°C. overnight and filtered by suction.

The filter cake was washed with 50 mL of icy ethyl acetate to give 6.66g of a yellow-green solid with a yield of 65.09%.

Example 16

Preparation of a monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionewith high purity

2.0 g of the crude product disclosed in Example 1 was placed in a brownbottle, 125 mL of isopropanol was added under heating till said crudeproduct completely dissolved. 50 mL of water was added resulting in nocrystalline precipitation. The resulting solution was stirred and cooledto precipitate at room temperature and filtered by suction. Theresulting filter cake was washed with a 1:1 mixture of isopropanol andwater and dried to give 1.642 g of a yellow powdery solid with a yieldof 78.13%. The product purity at 254 nm is 99.94%, wherein the contentof isomer(3E,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionewas 0.06%, see FIG. 18. ¹H NMR (500 MHz, dmso) δ12.22 (brs,2H), 10.00(brs, 1H), 7.82 (d, J=12.7 Hz, 1H), 7.51 (d, J=7.6 Hz, 2H), 7.40 (t,J=7.7 Hz, 2H), 7.30 (t, J=7.4 Hz, 1H), 6.73 (s, 1H), 1.37 (s, 9H), MS(ESI) m/z 338.1715 (M+H)⁺ (calcd for C₁₉H₂₀DN₄O₂).

The product obtained is a monohydrate of(3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazol-4-yl)deuteromethylene)piperazine-2,5-dione,containing 5.314% water. FIG. 19 shows the supporting data provided by athermogravimetric analysis, wherein the element analysis is shown inTable 4 below.

Example 17

Preparation of a crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

The specific preparation process includes the following steps:

1) Preparation of 5-(tert-butyl)oxazole-4-ethyl formate 90 g (796 mmol)of ethyl isocyanoacetate was added to 1000 mL of tetrahydrofuran,followed by slow dropwise addition of 145 g (955 mmol) of DBU anddropwise addition of 178 g (955 mmol) of trimethylacetic anhydride. Thereaction was stirred at room temperature for 48 hours. After thereaction was completed, the mixture was concentrated under reducedpressure. Extracted, an appropriate amount of 1500 mL of dichloromethanewas added, followed by successive washing with 800 mL of 10% sodiumcarbonate, 800 mL of 10% citric acid and 800 mL of saturated brine. Theaqueous phase was back-extracted twice with 1000 mL of dichloromethane.The organic phases were combined, dried over anhydrous sodium sulfatefor half an hour, filtered by suction, concentrated under reducedpressure and filtered through silica gel (200-300 mesh) column (EA:PE=1:10, 1:8, 1:5) to give 177 g of 5-(tert-butyl)oxazole-4-ethylformate as a yellow liquid with a yield of 113%.

2) Preparation of 5-(tert-butyl)-1H-imidazole-4-ethyl formate 157 g (796mmol) of 5-(tert-butyl)-1H-oxazole-4-ethyl formate was added to 717 g(15.914 mmol) of formamide. The mixture was let stand in an oil bath at180° C., stirred under reflux for 30 hours, cooled to room temperature,extracted and added with 800 mL of 10% sodium carbonate. 500 mL ofpetroleum ether was added to extract and the organic phase wasdiscarded. Then extracted three times with ethyl acetate (1000 mL*3).The organic phases were combined and washed twice with saturated brine(800 mL*2), The aqueous phase was back-extracted twice with ethylacetate (500 mL*2). The organic phases were combined and dried overanhydrous sodium sulfate, filtered by suction, concentrated underreduced pressure, pulpified, added with 1000 mL of water, stirred andfiltered by suction. The resulting filter cake was washed with water anddried in vacuo at 50° C. to give 71 g of said5-(tert-butyl)-1H-imidazole-4-ethyl formate as a earthy-yellow solidwith a yield of 45%.

3) Preparation of 5-(tert-butyl)-1H-imidazole-4-methanol

40 g (1054 mmol) of lithium aluminum hydride was added to 300 mL ofdried tetrahydrofuran in a cold trap at −10° C. 70 g (357 mmol) of5-(tert-butyl)-1H-imidazole-4-ethyl formate dissolved in 200 mL oftetrahydrofuran was slowly added dropwise to the turbid solution oflithium aluminum hydride. The reaction was stirred at room temperaturefor 3 hours. Quenched by ice-water, the reaction solution was addeddropwise into a measuring cup with an appropriate amount of ice, andfiltered by suction. The resulting filter cake was successively washedtwice with water (1000 mL * 2), twice with tetrahydrofuran (500 mL * 2),twice with anhydrous ethanol (500 mL * 2), concentrated under reducedpressure, and dehydrated by anhydrous ethanol to give 51 g of said5-(tert-butyl)-1H-imidazole-4-methanol as a pale-yellow solid with ayield of 93%.

4) Preparation of 5-(tert-butyl)-1H-imidazole-4-carbaldehyde

50 g (324 mmol) of 5-(tert-butyl)-1H-imidazole-4-methanol was added to500 mL of dichloromethane, followed by addition of 282 g (3242 mmol) ofmanganese dioxide. The reaction was stirred at room temperature for 24hours. The reaction mixture was filtered by suction and added withdiatomite. The resulting filter cake was washed three times withanhydrous ethanol (500 mL*3) and concentrated under reduced pressure togive 43 g of said 5-(tert-butyl)-1H-imidazole-4-carbaldehyde as apale-yellow solid with a yield of 87%.

5) Preparation of N,N-diacetylpiperazine-2,5-dione

50 g (438 trawl) of glycine anhydride was added to 179 g (1753 mmol) ofacetic anhydride. The mixture was let stand in an oil bath at 155° C.,stirred under reflux for 30 hours, concentrated under reduced pressure,dissolved in dichloromethane and filtered through diatomite and silicagel. The filter cake was rinsed with dichloromethane, concentrated underreduced pressure, dissolved in ethyl acetate at 70° C. andrecrystallized to give 74 g of said N,N-diacetylpiperazine-2,5-dione asa brown solid with a yield of 85%.

6) Preparation of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

1 g (6.5 mmol) of 5-(tert-butyl)-1H-imidazole-4-carbaldelryde was addedto 7 mL of DMF, followed by addition of 2.59 g (13 mmol) ofN,N-diacetylpiperazine-2,5-dione. The resulting solution was repeatedlyexhausted under nitrogen for three times. 3.19 g (9.8 mmol) of cesiumcarbonate was added and the mixture was repeatedly exhausted undernitrogen for three times. The reaction was stirred at room temperaturefor 20 hours in the absence of light. The reaction solution was pouredinto ice-water (100 mL) and filtered by suction. The resulting filtercake was successively washed with water (100mL*2) and an 8:1 mixture ofpetroleum ether and ethyl acetate (90 mL), then ultrasonically dispersedin ethanol and dichloromethane. The insolubles was filtered off and theresulting filtrate was concentrated under reduced pressure, dehydratedwith anhydrous ethanol, and pulpified with ethyl acetate (50 mL) to give0.89 g of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneas a brown-yellow solid with a yield of 46.9%.

7) Preparation of a crude product of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione

0.85 g (2.9 mmol) of(Z)-1-acetyl-3-[(5-(tert-butyl)-1H-imidazol-4-yl)methylene]piperazine-2,5-dione was added to DMF, followed by addition of 0.56 g(5.25 mmol) of benzaldehyde. The resulting solution was repeatedlyexhausted under nitrogen for three times. 0.95 g (2.9 mmol) of cesiumcarbonate was added and the mixture was repeatedly exhausted undernitrogen for three times. The temperature was programmed to reach 80° C.and the reaction was stirred for 24 hours in the absence of light. Thereaction solution was poured into ice-water (100 mL) and filtered bysuction. The resulting filter cake was washed successively with water(100 mL*2) and an 8:1 mixture of petroleum ether and ethyl acetate (90mL), then untrasonically dispersed in ethanol (30 mL) and ethyl acetate(100 mL). The insolubles was filtered off and the resulting filtrate wasconcentrated under reduced pressure, dehydrated with anhydrous ethanol,dispersed in 50 mL of ethyl acetate, let stand at −30° C. overnight andfiltered by sanction. The resulting filter cake was then washed with 5mL of icy ethyl acetate to give 0.73 g of a crude product

Example 18

Preparation of a monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewith high purity

0.73 g of said crude product was placed in a brown bottle, 45 mL ofisopropanol was added under heating till said crude product completelydissolved. 18 mL of water was added resulting in no crystallineprecipitation. The resulting solution was stirred and cooled toprecipitate at room temperature and filtered by suction. The resultingfilter cake was washed with a 1:1 mixture of isopropanol and water, anddried to give 0.59 g of a yellow powdery solid with a yield of 76.71%.The product purity was 99.91% at 254 nm, wherein the content of isomer(3E,6Z)-3-benzylidene-6[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dionewas 0.09%, as shown in FIG. 20; ¹H NMR (400 MHz, DMSO-d6) δ 12.31 (s,1H), 12.22 (s, 1H), 10.00 (s, 1H), 7.84 (s, 1H), 7.52 (d, J=8 Hz, 2H),7.39 (t, J=8 Hz, 2H), 7.32 (t, J=8 Hz, 1H), 6.86 (s, 1H), 6.73 (s, 1H),1.37 (s, 9H); MS (ESI) m/z 337.1659 (M+H)⁺ (calcd for C₁₉H₂₁l N₄O₂).

The obtained product is a monohydrate of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-4-imidazol-4-yl)methylene]piperazine-2,5-dione,containing 5.326% water. FIG. 21 shows the supporting data provided by athermogravimetric analysis.

Example 19

Preparation of 5-(tert-butyl)-1H-imidazole-4-ethyl formate intermediate

157 g (796 mmol) of 5-(tert-butyl)-1H-oxazole-4-ethyl formate was addedto 717 g (15.91 mmol) of formamide. The mixture was let stand in an oilbath at 180° C., stirred under reflux for 30 hours, cooled to roomtemperature, extracted and added with 800 2.0 mL of 10% sodiumcarbonate. 500 mL of petroleum ether was added to extract and theorganic phase was discarded. Then extracted three times with ethylacetate (1000 mL*3). The organic phases were combined and washed twicewith saturated brine (800 mL*2). The aqueous phase was back-extractedtwice with ethyl acetate (500 mL*2). The organic phases were combinedand dried over anhydrous sodium sulfate, filtered by suction,concentrated under reduced pressure, pulpified, added with 1000 mLwater, stirred and filtered by suction. The resulting filter cake waswashed with water, dried in vacuo at 50° C. to give 71 g of5-(tert-butyl)-1H-imidazole-4-ethyl formate as a earthy-yellow solidwith a yield of 45%.

Example 20

Preparation of N,N-diacetylpiperazine-2,5-dione intermediate

50 g (438 mmol) of glycine anhydride was added to 179 g (1753 mmol) ofacetic anhydride. The mixture was let stand in an oil bath at 155° C.,stirred under reflux for 30 hours, concentrated under reduced pressure,dissolved in dichloromethane and filtered through diatomite and silicagel. The resulting filter cake was rinsed with dichloromethane,concentrated under reduced pressure, dissolved in ethyl acetate at 70°C. and recrystallized to give 74 g of N,N-diacetylpiperazine-2,5-dioneas a brown solid with a yield of 85%.

It should be noted that the preparation method provided by the presentinvention can not only be used for preparing a(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl)-1H-imidazol-4-yl)deuteromethylene]piperazine-2,5-dionemonohydrate with high purity and a(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl)-1H-imidazol-4-yl)methylene]piperazine-2,5-dione monohydrate with high purity as disclosed in the embodiments, butalso suitable for preparation and purification of alldehydrophenylahistin-like compounds with similar structures, which couldbe any dehydrophenylahistin-like compound disclosed in the patents ofWO2001053290A1, WO2004054498A, WO2007035841A1 or WO2016192586A1, orderivatives of these compounds. Preferably, thedehydrophenylahistin-like compounds disclosed herein have a structurerepresented by the following formula:

wherein R₁ is mono-substituted to penta-substituted substituent group onthe benzene ring, which is independently selected from hydrogen,deuterium, 3-benzoylphenyl, 3-(4-methoxybenzoyl)phenyl,3-(4-fluorobenzoyl)phenyl, halogen, hydroxy, methoxy, amino, phenyl,aminomethylphenyl, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl,arylalkyl, heterocycloarylalkyl, C1-C24 acyl, Cl-C24 alkoxy, carboxy,carboxy late, acylamino, N-monosubstituted or N,N-disubstitutedacylamino, sulfo, sulphonate, sulphonylamino, N-substitutedsulphonylamino, alkoxy, arylalkoxy, alkylthio, cyano, amino, substitutedamino, nitro, cycloalkyl, cycloalkenyl, aryl, substituted aryl,heterocycloaryl, arylox,z, aroyl, epoxy group, cycloacyl, arylthio,arylst lfonyl,

R₂ is hydrogen or deuterium, and R₃ is hydrogen or deuterium;

X₁ is oxygen or sulfur, and X₂ is oxygen or sulfur;

X₃ is -NH oxygen or sulfur.

In the structure of dehydrophenylahistin-like compounds disclosed hereinexists double bonds capable of forming a cis-trans isomer, and cis-transisomerization is highly likely to occur under illumination condition.The method for preparing a dehydrophenylahistin-like compound with highpurity disclosed in the claims of the present invention results fromoptimization of the purification methods for various similar compoundsin the present invention, which significantly reduces the impuritycontent of isomers and yields an active compound with its purity higherthan 99.9%.

Although detailed description of embodiments of the present inventionhas been described for the purpose of examplification, it is apparent tothose skilled in the art that various changes and modifications to thepreferred embodiments described herein can be made without departingfrom the spirit and scope of the present invention covered by theclaims.

1. (canceled)
 2. (canceled)
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 5. (canceled) 6.(canceled)
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 10. (canceled) 11.(canceled)
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 15. (canceled)16. A crystalline form of(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dioneas shown in Formula (II), wherein the crystalline form is selected fromthe group consisting of b-crystalline form, c-crystalline form andd-crystalline form, wherein the b-crystalline form has at least threeX-ray powder diffraction characteristic peaks at 2θ diffraction angle of7.670°±0.2°, 9.069°±0.2°, 15.383°±0.2°, 16.668°±0.2°, 17.468±0.2°,18.109°±0.2°, 19.960°±0.2°, 23.307°±0.2°, 23.836°±0.2°, 24.462°±0.2°,28.046°±0.2° or 28.827°±0.2°; the c-crystalline form has at least threeX-ray powder diffraction characteristic peaks at 2θ diffractionangle of7.918°±0.2°, 9.168°±0.2°, 12.014°±0.2°, 12.985°±0.2°, 18.382°±0.2°,18.616°±0.2°, 23.367°±0.2°, 25.203°±0.2° or 27.771°±0.2°; thed-crystalline form has at least three X-ray powder diffractioncharacteristic peaks at 2θ diffraction angle of 8.073°±0.2°,13.005°±0.2°, 17.544°±0.2°, 18.382°±0.2°, 19.082°±0.2°, 19.707°±0.2°,22.766°±0.2°, 23.759°±0.2°, 24.438°±0.2°, 25.277°±0.2°, 26.486°±0.2° or29.234°±0.2 ^(.),


17. The crystalline form according to claim 16, wherein the X-ray powderdiffraction pattern of the b-crystalline form is consistent with FIG.11.
 18. (canceled)
 19. The crystalline form according to claim 16,wherein the X-ray powder diffraction pattern of the c-crystalline formis consistent with FIG.
 13. 20. (canceled)
 21. The crystalline formaccording to claim 16, wherein the d-crystalline form is a monohydrateof(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione.22. The crystalline form according to claim 16, wherein the X-ray powderdiffraction pattern of the d-crystalline form is consistent with FIG.15.
 23. A method of preparing the d-crystalline form comprising thefollowing steps: using a mixed solvent of water and an organic solventas crystallization solvent, wherein the organic solvent is selected fromat least one of an alkane saturated alcohol, an unsaturated alcohol, analkane saturated amine and an unsaturated amine; recrystalling(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazol-4-yl)methylene]piperazine-2,5-dione in the mixed solvent, cooling and stirring to precipitate toobtain the d-crystalline form.
 24. The method according to claim 23,wherein the mixed solvent comprises water and isopropanol with a volumeratio of V_(water):V_(isopropanol) ranging from 1:100 to 99:100.
 25. Themethod according to claim 24, wherein the mixed solvent has a volumeratio of V_(water):V_(isopropanol) ranging from 1:10 to 9:10.
 26. Themethod according to claim 23, wherein the method comprises the followingsteps: placing(3Z,6Z)-3-benzylidene-6-[(5-tert-butyl-1H-imidazole-4-yl)methylene]piperazine-2,5-dionein a reaction vessel in the absence of light, adding isopropanol assolvent, heating till dissolving completely, then adding water with avolume ratio of isopropanol to water ranging from 1:10 to 100:1, keepingat −15° C. to 30° C., stirring and cooling to precipitate, suctionfiltering, washing and drying to obtain the d-crystalline form.
 27. Themethod according to claim 26, wherein the volume ratio of isopropanol towater is 5:2, preferably keeping at −5° C. to 10° C., stirring andcooling to precipitate.
 28. (canceled)